Ubiquitin is a small protein which functions at the level of protein regulation. Proteins are post-translationally modified by covalent conjugation with ubiquitin in a process referred to as ubiquitination. Among the regulatory functions of ubiquitin, polyubiquitination can mark a modified protein for proteasome-mediated degradation.
Deubiquitinase proteins play significant roles in cell-cycle regulation, signaling, DNA repair, chromatin remodeling, and other important biological functions. Deubiquitinase proteins serve to catalytically hydrolyze ubiquitin moieties from ubiquitinated proteins. In many cases, ubiquitination serves as a flag for delivery to the proteasome. Removal of ubiquitin moieties prior to interaction of the substrate with the proteasome can stave off degradation of that substrate. Notably, some deubiquitinases associate directly with the proteasome, and function to remove the polyubiquitin signal to permit completion of substrate degradation. In this way, deubiquitinases can either positively or negatively regulate degradation of proteasomal substrates.
Activity of the proteasome is deregulated in numerous human cancers. The human proteasomal Rpn11 deubiquitinase (also known as PSMD14, POH1. S13, Pad1, Mpr1) is a validated target for anticancer therapy. However, identification of potent, selective inhibitors has been thwarted by its poor in vitro activity and the lack of target-specific cell-based assays.
Cell-based assays which report directly on Rpn11 activity are currently unavailable. While biochemical screening methodologies have been previously employed, they require expensive reagents and do not address critical parameters necessary for further drug development. For instance, biochemical screens fail to address cell permeability, acute cellular toxicity, and other important factors required for identifying compounds useful for treating Rpn11-related diseases.
Thus, there is a need to address the aforementioned problems and other shortcomings associated with identification of anti-deubiquitinase compounds.